A versatile viral toolkit for functional discovery in the nervous system.
Ontology highlight
ABSTRACT:
SUBMITTER: Pouchelon G
PROVIDER: S-EPMC9243523 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundRice (Oryza sativa) is the main food for half of the world's population, and is considered the model for molecular biology studies of monocotyledon species. Although the rice genome was completely sequenced about 15 years ago, the function of most rice genes is still unknown.ResultsIn this study, we developed a vector toolkit that contains 42 vectors for transient expression studies in rice protoplasts and stable expression analysis in transgenic rice. These vectors have been successfully used to study protein subcellular localization, protein-protein interaction, gene overexpression, and the CRISPR/Cas9-mediated gene editing. A novel feature of these vectors is that they contain a universal multiple cloning site, which enables more than 99% of the rice coding sequences to be conveniently transferred between vectors.ConclusionsThe versatile vectors represent a highly efficient and high-throughput toolkit for functional analysis of rice genes.
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Project description:BackgroundThe ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies.ResultsAiming at high efficiency and high throughput methods, we present here a construction kit for gene knock out in T. reesei. We provide a primer database for gene deletion using the pyr4, amdS and hph selection markers. For high throughput generation of gene knock outs, we constructed vectors using yeast mediated recombination and then transformed a T. reesei strain deficient in non-homologous end joining (NHEJ) by spore electroporation. This NHEJ-defect was subsequently removed by crossing of mutants with a sexually competent strain derived from the parental strain, QM9414.ConclusionsUsing this strategy and the materials provided, high throughput gene deletion in T. reesei becomes feasible. Moreover, with the application of sexual development, the NHEJ-defect can be removed efficiently and without the need for additional selection markers. The same advantages apply for the construction of multiple mutants by crossing of strains with different gene deletions, which is now possible with considerably less hands-on time and minimal screening effort compared to a transformation approach. Consequently this toolkit can considerably boost research towards efficient exploitation of the resources of T. reesei for cellulase expression and hence second generation biofuel production.
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Project description:The caption of Fig. 5 and the figure contained an error [1]. The updated figure and caption are published in this correction article.
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Project description:Tissue clearing combined with deep imaging has emerged as a powerful alternative to classical histological techniques. Whereas current techniques have been optimized for imaging selected nonpigmented organs such as the mammalian brain, natural pigmentation remains challenging for most other biological specimens of larger volume. We have developed a fast DEpigmEntation-Plus-Clearing method (DEEP-Clear) that is easily incorporated in existing workflows and combines whole system labeling with a spectrum of detection techniques, ranging from immunohistochemistry to RNA in situ hybridization, labeling of proliferative cells (EdU labeling) and visualization of transgenic markers. With light-sheet imaging of whole animals and detailed confocal studies on pigmented organs, we provide unprecedented insight into eyes, whole nervous systems, and subcellular structures in animal models ranging from worms and squids to axolotls and zebrafish. DEEP-Clear thus paves the way for the exploration of species-rich clades and developmental stages that are largely inaccessible by regular imaging approaches.
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Project description:Developing drugs for the central nervous system (CNS) requires fine chemical modifications, as a strict balance between size and lipophilicity is necessary to improve the permeability through the blood-brain barrier (BBB). In this context, morpholine and its analogues represent valuable heterocycles, due to their conformational and physicochemical properties. In fact, the presence of a weak basic nitrogen atom and of an oxygen atom at the opposite position provides a peculiar pKa value and a flexible conformation to the ring, thus allowing it to take part in several lipophilic-hydrophilic interactions, and to improve blood solubility and brain permeability of the overall structure. In CNS-active compounds, morpholines are used (1) to enhance the potency through molecular interactions, (2) to act as a scaffold directing the appendages in the correct position, and (3) to modulate pharmacokinetic/pharmacodynamic (PK/PD) properties. In this perspective, selected morpholine-containing CNS drug candidates are discussed to reveal the active pharmacophores accountable for the (1) modulation of receptors involved in mood disorders and pain, (2) bioactivity toward enzymes and receptors responsible for neurodegenerative diseases, and (3) inhibition of enzymes involved in the pathology of CNS tumors. The medicinal chemistry/pharmacological activity of morpholine derivatives is discussed, in the effort to highlight the importance of morpholine ring interactions in the active site of different targets, particularly reporting binding features retrieved from PDB data, when available.
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Project description:Recombinant adeno-associated virus (AAV) is a promising delivery vehicle for in vivo gene therapy and has been widely used in >200 clinical trials globally. There are already several approved gene therapy products, e.g., Luxturna and Zolgensma, highlighting the remarkable potential of AAV delivery. In the past, AAV has been seen as a relatively non-immunogenic vector associated with low risk of toxicity. However, an increasing number of recent studies indicate that immune responses against AAV and transgene products could be the bottleneck of AAV gene therapy. In clinical studies, pre-existing antibodies against AAV capsids exclude many patients from receiving the treatment as there is high prevalence of antibodies among humans. Moreover, immune response could lead to loss of efficacy over time and severe toxicity, manifested as liver enzyme elevations, kidney injury, and thrombocytopenia, resulting in deaths of non-human primates and patients. Therefore, extensive efforts have been attempted to address these issues, including capsid engineering, plasmapheresis, IgG proteases, CpG depletion, empty capsid decoy, exosome encapsulation, capsid variant switch, induction of regulatory T cells, and immunosuppressants. This review will discuss these methods in detail and highlight important milestones along the way.
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Project description:The ability of the central nervous system (CNS) to generate innate immune responses was investigated in an in vitro model of CNS infection. Cultures containing CNS cells were infected with mouse hepatitis virus-JHM, which causes fatal encephalitis in mice. Immunostaining indicated that viral infection had a limited effect on culture characteristics, overall cell survival, or cell morphology at the early postinfection times studied. Results from Affymetrix gene array analysis, assessed on RNA isolated from virally and sham-infected cultures, were compared with parallel protein assays for cytokine, chemokine, and cell surface markers. Of the 126 transcripts found to be differentially expressed between viral and sham infections, the majority were related to immunological responses. Virally induced increases in interleukin-6 and tumor necrosis factor alpha mRNA and protein expression correlated with the genomic induction of acute-phase proteins. Genomic and protein analysis indicated that viral infection resulted in prominent expression of neutrophil and macrophage chemotactic proteins. In addition, mRNA expression of nonclassical class I molecules H2-T10, -T17, -M2, and -Q10, were enhanced three- to fivefold in virus-infected cells compared to sham-infected cells. Thus, upon infection, resident brain cells induced a breadth of innate immune responses that could be vital in directing the outcome of the infection and, in vivo, would provide signals which would summon the peripheral immune system to respond to the infection. Further understanding of how these innate responses participate in immune protection or immunopathology in the CNS will be critical in efforts to intervene in severe encephalitis.
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Project description:Circular RNAs (circRNAs) are a class of ubiquitously expressed, single-stranded, covalently covalently-closed (i.e. circularised) RNA that contain a unique nucleotide sequence created by the ligation of their 5' and 3' ends, called the back-splice junction. Understanding the cellular roles of circRNAs involves, in part, investigating the effects on cell phenotype of increased expression of individual circRNAs. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote circularisation. We observed that all commonly used plasmids we tested unexpectedly incorporated molecular scars comprising vector sequence vector sequence as a molecular scar into the circRNA back-splice junction upon circularisation. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural back-splice junction at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that enable selection with a variety of antibiotics and fluorescent proteins, a range of promoters varying in promoter strength and plus a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfil a critical need for the investigation of circRNA function, including the role of the unique back-splice junction.
2023-12-14 | GSE246020 | GEO
Project description:MotivationConverting nucleotide sequences into short overlapping fragments of uniform length, k-mers, is a common step in many bioinformatics applications. While existing software packages count k-mers, few are optimized for speed, offer an application programming interface (API), a graphical interface or contain features that make it extensible and maintainable. We designed KAnalyze to compete with the fastest k-mer counters, to produce reliable output and to support future development efforts through well-architected, documented and testable code. Currently, KAnalyze can output k-mer counts in a sorted tab-delimited file or stream k-mers as they are read. KAnalyze can process large datasets with 2 GB of memory. This project is implemented in Java 7, and the command line interface (CLI) is designed to integrate into pipelines written in any language.ResultsAs a k-mer counter, KAnalyze outperforms Jellyfish, DSK and a pipeline built on Perl and Linux utilities. Through extensive unit and system testing, we have verified that KAnalyze produces the correct k-mer counts over multiple datasets and k-mer sizes.Availability and implementationKAnalyze is available on SourceForge: https://sourceforge.net/projects/kanalyze/.
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